Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique.We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50–60 nm on a time scale of 2.3 s.Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level. In the nucleus of eukaryotic cells, chromatin is organized into two distinct domains: the lightly packed and actively transcribed euchromatin, and the condensed and transcriptionally silent heterochromatin. Although transcriptionally repressive, heterochromatin is known to provide binding sites for regulatory proteins, and therefore, plays an important role in the epigenetic regulation of a variety of biological processes, such as development, cellular differentiation and maintenance of genome integrity . Human eon par with the electron micrograph , even with the aid of the light-sheet illumination. Since the Nyquist sampling theorem requires approximately two data points per resolution unit to faithfully reconstruct an image , also known as light-sheet microscopy, has been recently introduced for single-molecule super-resolution imaging by Zanacchi et al . The plaet al . To geneet al . For imaet al . Consideet al  and inveet al   and focused onto the back focal plane of an infinity-corrected Mitutoto M Plan APO 50× NA 0.55 condenser objective. A Pellin-Broca prism (Thorlabs ADBU-10) was placed after the condenser objective to redirect the light sheet horizontally onto a sample at the focal plane of an imaging objective, in our case, a Zeiss W Plan-Apochromat 63× NA 1.0 water immersion lens.To adapt the light sheet illumination for imaging single hESCs, we have developed a simple and effective prism-coupled light-sheet condenser design. Demonstrated in The prism-coupled condenser design enjoys three major advantages for super-resolution imaging. First, the de-coupling between the imaging and condenser objective and increase of the angle between them from 90 to roughly 120 degrees relaxes the space constraint for sample placement. The design widens the choice of imaging objectives and allows the use of even higher NA (NA > 1.0) water-immersion lenses. Second, the horizontal overlap between light-sheet illumination and the imaging plane yields a flat and large FOV with high SNR. Large FOV is necessary in order to capture the entire cross-section of the nucleus of the hESC. Third, compared to the AFM cantilever system , the prir is the radial distance from the center of the beam, z is the axial distance, w0 is the beam waist and set to 2 µm, 2e, z. We plotted the intensity as the square of the normal E field. The FWHM from the line plot of the intensity profile characterized the width and length of the light sheet. For demonstration, we simulated the light sheet at 561 nm. The refractive index was 1.333 for water and 1.512 for glass.We used the RF module in COMSOL Multiphysics™ to perform the full-wave electromagnetic simulation of the light sheet. The incident Gaussian beam was characterized in the form:6× dilution. A glass bottom dish containing 3 ml diluted fluorescent bead solution was use for profiling. The thickness was determined from the image captured by the EMCCD camera and the image pixel size.For visualizing and measuring the thickness of the light sheet, we used fluorescent bead solution (Invitrogen F-8794) with 10Human embryonic stem cells were cultured at the Stem Cell Core facility of the Salk Institute as previously described . Two NIHFor immunostaining, we followed standard labeling procedures for STORM. Briefly, cells were fixed in 3% (v/v) paraformaldehyde (Alfa Aesar 43368) and 0.1% glutaraldehyde (Sigma G5882) (v/v) in PBS for 10 min and washed three times in filtered PBS. The remaining PFA and glutaraldehyde were reduced by incubating fixed cells in freshly prepared 0.1% (w/v) sodium borohydride (Sigma 213462) for 7 min, followed by permeabilization in the blocking buffer (5% (v/v) normal goat serum and 0.2% (v/v) Triton X100 (Sigma T8787) in PBS) for approximately one hour. The primary mouse anti-β-tubulin antibody (Invitrogen 1083461A) was diluted 200× in the blocking buffer with a 30-min incubation time. After washing five times in the washing buffer (0.2% (v/v) normal goat serum and 0.1% (v/v) Triton x100 in PBS), cells were incubated in the Alexa Fluor 647 conjugated goat anti-mouse IgG secondary antibody (Moleular Probes A21237) with 500× dilution in the blocking buffer for 30 min. The sample was protected from light and post-fixed with 3% (v/v) paraformaldehyde and 0.1% (v/v) glutaraldehyde in PBS for 10min after three-time wash.−1 glucose oxidase (Sigma G6125), 40 µg ml−1 catalase (Sigma C1345), and 143 mM β-mercaptoethanol (Sigma M6250) at pH 8.0. For vertical sectioning, the sample was translated tangential to the top surface of the prism by the piezo stage. With a known tilt angle, vertical translates can be precisely determined from the tangential movement.The image frame size was cropped to smaller than 128×128 pixels in order to increase the frame rate. Low excitation power was used to first locate the nucleus or microtubules, followed by imaging with full excitation power. The initial quick bleaching and subsequent blinking process were typically captured within the first 1–2000 frames with an exposure time of 30–50 ms. Standard imaging buffer was used for STORM: 40 mM D-glucose, 0.5 mg mlThe light-sheet microscope was adapted for short-duration live-cell imaging. Temperature was maintained by heating the imaging objective lens and glass prism using objective heaters . The imaging duration was less than 30 min. Transgenic Hues6 cells expressing mEos3-HP1α were seeded on glass bottom dishes with phenol-red free TeSR 30 min prior to imaging. For 250 ml of colorless TeSR, 500 µl of FGF-2, and 30 µl of TGFb1 were added to prevent cell differentiation. To minimize adverse phototoxicity, 1mW 405 nm laser was used to activate mEos3 for 5 s, followed by bleach-and-blink acquisition using the 561-nm laser at 40 mW for ~23 s with 4000 frames. Approximately 26 mW from the 561-nm laser and 200 µW from the 450-nm laser were delivered to the back aperture of the light sheet condenser objective. The exposure time was 50 ms and the frame size was 75×89 pixels. For imaging fixed cells, we used a custom-designed system and paired a 300- mm tube lens with a 63× Zeiss objective. The effective magnification was 114×, yielding a pixel size of 135 nm. For live cell imaging, we coupled light sheet into a commercial Ziess microscope with a 63× magnification, yielding a pixel size of 254 nm. A 2.5× magnifier can be used to further reduce the pixel size to 102 nm.et al.  and size (2R = 0.9957) values of the single-molecule events  service for labs without the access to supercomputing resources  Center at the Lawrence Berkeley National Laboratory. For image reconstruction, over-exposed images at the beginning of the acquisition were discarded and subsequent 400 images (saved as fits files) were selected for processing. The 3B analysis was parallelized by dividing the image into 10-by-10-pixel sub-masks, each with a 50% horizontal and vertical overlap with its neighbors to eliminate edge artifacts. A Python-MPI wrapper was used to distribute the Bayesian calculation to a number of processors equal to the number of sub- masks. For a 128-by-128-pixel image, approximately 2,500 CPU hours were consumed. Details of the Bayesian algorithm and its implementation are discussed in esources .w0, and its length b determined by rd order aberrations, such as coma, induced at the interface, as well as the refractive index of the imaging medium (We used finite-element simulations to help tailor the design parameters for imaging the hESC nucleus, which is ~10-µm thick and wide. As a first-order approximation, the thickness of the light sheet is determined by the diffraction-limited waist size of a focused Gaussian beam, he prism  and founhe prism . By turnhe prism , we meashe prism , which ig medium . In addiThe enhanced SNR using light sheet illumination can be observed from single-molecule images. As a comparison, we performed single-molecule imaging of fixed mEos3-HP1α-labeled transgenic Hues6 hESCs using high-angle epi and prism-coupled light sheet illumination . Figure The Bayesian algorithm effectively resolves sub-diffraction cellular structures. We cross-compared the reconstructed structures between Bayesian and standard single-emitter fitting analysis using standard epi and light-sheet illumination for both thin and thick cell samples. Albeit substantial reduced number of image frames, the Bayesian analysis can achieve comparable resolution to standard PALM/STORM techniques. We generated transgenic hESC cell lines expressing photoactivatable proteins mEos2 or mEos3 on the heterochromatin protein HP1α using lenti-viral infection. Known to be enriched at the heterochromatic sites and maintaining the stability of heterochromatin, HP1α is regarded as a hallmark of heterochromatin . We perfvia a piezoelectric stage (The pattern of HP1α distribution was consistently found at various depths of the nucleus . By utilic stage . As the With the enhanced temporal resolution, we demonstrate live-cell imaging of heterochromatin in hESCs using LSBM. We adapted the LSBM for live-cell imaging by maintaining the temperature of the glass prism and the imaging objective . We conti.e. by using a cropped mode for a smaller FOV, can further increase the imaging speed to the sub-second level.Acquiring sub-cellular super-resolution images deep inside a living cell is one of the most challenging tasks in bioimaging. In this study, we designed LSBM and demonstrated its capability by visualizing the structure and dynamics of heterochromatin in fixed and live hESCs. With the fixed hESCs, we first demonstrated a mesh like heterochromatin structure in pluripotent stem cells. These structurally distinct organizations are also aligned with the topological domain hypothesis . The latin vivo studies from deep-cell imaging of sub-cellular structures to real-time probing of dynamic cellular processes with nanometer resolution.Our prism-coupled light sheet design provides high SNR for imaging thick hESCs and accommodates a high-NA imaging objective, which is crucial for the photon collection efficiency for single-molecule super-resolution imaging. The majority of light sheet designs use an imaging lens with an NA of 0.8 or lower. However, a mere increase of 0.2 in NA from 0.8 to 1.0 increases the light collection efficiency by 32%. Although we demonstrated single-molecule super-resolution imaging through 10-µm thick nuclei in hESCs, the use of LSBM can be readily expanded for general biological samples more than a few micrometers thick, such as thin tissue slices, without modification. In fact, the robustness and ease of implementation is a significant advantage over other light-sheet techniques. Taken together, LSMB enables a wide array of


Background: Exercise training improves performance and biochemical parameters on average, but wide interindividual variability exists, with individuals classified as responders (R) or non-responders (NRs), especially between populations with higher or lower levels of insulin resistance. This study assessed the effects of high-intensity interval training (HIIT) and the prevalence of NRs in adult women with higher and lower levels of insulin resistance.Methods: Forty adult women were assigned to a HIIT program, and after training were analyzed in two groups; a group with higher insulin resistance  and a group with lower insulin resistance . Anthropometric, cardiovascular, metabolic, and performance variables were measured at baseline and after 10 weeks of training.Results: There were significant training-induced changes [delta percent (Δ%)] in fasting glucose, fasting insulin, and homeostasis model assessment of insulin resistance (HOMA-IR) scores in the H-IR group , whereas no significant changes were observed in the L-IR. Both groups showed significant pre-post changes in other anthropometric variables . Systolic blood pressure decreased significantly only in the L-IR group . Both groups showed significant increases in 1RMLE . There were significant differences in the prevalence of NRs between the H-IR and L-IR groups for fasting glucose  and fasting insulin (p = 0.025) but not for HOMA-IR .Conclusion: Independent of the “magnitude” of the cardiometabolic disease , no differences were observed in the NRs prevalence with regard to improved HOMA-IR or to anthropometric, cardiovascular, and muscle performance co-variables after 10 weeks of HIIT in sedentary adult women. This research demonstrates the protective effect of HIIT against cardiometabolic disease progression in a sedentary population. Exercise training is a strategy for the prevention and treatment of several inactivity-related metabolic diseases, such as insulin resistance , has been reported predominantly after endurance training  with no background of regular exercise training volunteered to be screened.n = 65) were excluded for multiple reasons . Similarly, 25 subjects identified as “lower insulin-resistance” subjects (n = 25) were also excluded for similar reasons . Finally, 78 screened subjects with a higher level of insulin resistance (n = 78) were assigned to 10 weeks of a HIIT program and were analyzed after intervention in two different groups: a group with a higher level of insulin resistance  and a group with a lower level of insulin resistance . None of the subjects were taking oral hypoglycemic medications to improve metabolic control of glucose because they all had been recently diagnosed with insulin resistance by our research team. Subjects with < 70% attendance at training sessions were excluded from all statistical analyses after the intervention; after excluding those subjects, the characteristics of the analyzed subject groups were as follows: H-IR group, age 40 ± 6 years, n = 20; L-IR group, age 35 ± 9 years, n = 20 . In the first screening before intervention, 65 individuals classified as “higher insulin-resistance” subjects . Characteristics of the study participants are provided in Tables Eligibility criteria included the following: (a) diagnosed with insulin resistance based on the HOMA-IR metabolic marker method and using a cut-off point of HOMA-IR ≥2.6 in a Chilean population  of the difference in scores observed between the two repeats of each test. A non-responders participant for HOMA-IR assessments, as well as for all other included co-variables, was defined as an individual who failed to demonstrate a decrease or increase  that was greater than two times the TE from zero. A change greater than two times the TE means that there is a high probability  that this response is a true physiological adaptation beyond what might be expected to result from technical and/or biological variability . Three skinfold measurements of subcutaneous adipose tissue  were assessed using a Langue™ skinfold caliper  according to standard protocols . Height was assessed with a professional stadiometer  with an accuracy of 0.1 cm, and body mass index (BMI) was calculated according to the following formula: kg/mSystolic and diastolic blood pressure were assessed using an automatic monitor  in triplicate  after 15 min of rest and with the subjects in a seated position following standard classification procedures  were collected in tubes with specific anticoagulant gels for fasting glucose and fasting insulin measurements at baseline and at the 10 week follow-up. Samples were placed on ice and centrifuged at 4,000 rpm  for 5 min at 4°C. Plasma samples were immediately transferred to pre-chilled microtubes and stored at −20°C for later analysis.Plasma glucose was analyzed via enzymatic methods using standard kits  on an automatic analyzer . Fasting insulin was measured via RIA . The HOMA-IR index was calculated using the Matthews equation , and in the 1RMUR-test, free weights with bars were used. In brief, for the 1RMLE-test, the subjects began by lifting a load of weights on the machine with both legs. For the 1RMUR-test, the subjects adopted a body flexion angle of 90°, grabbed a bar with weights and plates, and with both arms extended, lifted the bar to approximately knee height. The highest load from three attempts per exercise was reported.In week 5, after a familiarization process with the test and before the intervention, both groups performed one-repetition maximum strength tests to establish 1RMThe HIIT program was started in the sixth week and was performed three times per week, for a total of 30 sessions, using exercise bikes . Each participant performed a range of 8–12 cycling intervals during the intervention period. The time of each cycling work interval was 60 s, with 120 s of passive rest (sitiing on the bicycle without movement) between work intervals. This rest period was progressively decreased , reaching a time of 1.15 min in the tenth week. Cycle revolutions were maintained at a range of 50–70 rpm and a speed between 20 and 40 km/h during each work interval. Subjects were required to cycle at levels between 8 and 10 points on a modified 0–10 Borg scale during the work interval  was used to determine differences between categorical variables for R and NRs by group (H-IR × L-IR). After the intervention, the typical error (TE) were calculated for the pre-post changes for each dependent variable. The subjects were categorized as a R or NRs according to the previously described criteria of a change greater than two times the TE , with threshold values at 0.20, 0.60, 1.2, and 2.0 for small, moderate, large, and very large effects, respectively . Normality and homoscedasticity assumptions for all data were assessed using the Shapiro-Wilk-test and Levene's-test, respectively. The Wilcoxon-test was used for non-parametric data. One-way ANOVA was performed to test differences between groups at baseline. An ANCOVA was conducted to analyze variables that were significantly different at baseline. A repeated-measures ANOVA (group × time) was used to determine differences in all dependent variables between pre- and post-tests using each group × time. A chi-square test (p ≤ 0.05) differences between groups for abdominal skinfold thickness (Table p ≤ 0.05) pre-post changes [presented as delta percent (Δ%)] in waist circumference  and in tricipital , supra-iliac , and abdominal skinfold thicknesses  in both the H-IR and L-IR groups  differences between the groups for diastolic or systolic blood pressure  differences between the groups for fasting glucose, fasting insulin, and HOMA-IR scores (Table p ≤ 0.05) pre-post changes [presented as delta percent (Δ%)] in fasting glucose (−8.8%), fasting insulin (−26.5%), and HOMA-IR scores (−32.1%) in the H-IR group  differences between groups for 1RMLE and 1RMUR (Table p ≤ 0.05) pre-post changes in 1RMLE in the H-IR (+12.9) and L-IR (+14.7%) groups  in the L-IR group  and fasting insulin . There were no significant differences between the H-IR vs. L-IR groups in NRs prevalence with regard to a decrease in HOMA-IR scores , or cardiovascular parameters , muscle performance  associated with improved waist circumference , diastolic blood pressure , fasting glucose , and 1RMLE, 1RMUR) measures.The present study was designed to assess the effects 10 weeks of HIIT and NRs prevalence (as indicated by glucose control parameters) in adult women with higher and lower levels of insulin resistance to test if the “magnitude” of a metabolic disease play a role in increasing or decreasing the NRs prevalence. The major findings of this study indicate that (i) HIIT promotes significantly more benefits in training-induced changes in fasting glucose, fasting insulin and HOMA-IR scores in adult women with higher insulin resistance; (ii) the NRs prevalence was significantly different between the H-IR vs. L-IR groups with regard to improve fasting glucose and fasting insulin but not for HOMA-IR scores; and (iii) both the H-IR and L-IR groups experienced similar positive training-induced changes and similar NRs prevalence with regard to anthropometric , cardiovascular (systolic/diastolic blood pressure), and muscle strength performance  to tests the effect of frequency in older NRs subjects for 12 and 24 weeks. Major differences between both training regimens were found for body mass, which decreased by ~4.5% at 12 weeks and 23% at 24 weeks. Interestingly, other results included increases in type I (+34.5 vs. +29.4%) and type II muscle fibers (+22.7 vs. +21.1%), as well as increasing leg strength in extension exercises (+0.9 vs. +1.17%) at 12 and 24 weeks, with relatively similar results obtained independent of the training frequency. These results indicated that the frequency of training was not necessarily related to the NRs prevalence for some variables  for 20 weeks], 42% of subjects were NRs for a decrease in HOMA-IR scores. We found similar results regarding a decrease in HOMA-IR scores, with a NRs prevalence of 15 and 20% for the H-IR and L-IR groups, respectively  for 4 weeks].Understanding the NRs prevalence after exercise modes such as HIIT and including populations with higher and lower risks of T2DM, such as those with higher and lower levels of insulin resistance, can be useful for designing more efficient exercise interventions: in this case, populations with higher levels of insulin resistance, such as the H-IR group, defined based on fasting glucose and HOMA-IR scores, are less likely to be NRs after 10 weeks of HIIT. This altered baseline, which we termed previously as “higher insulin resistance,” may be in some way related to potential factors for predicting responses in future long-term studies. Collectively, and in combination with previous reports , RT , or concurrent training, approximately ~60% of subjects were NRs for a decrease in systolic and diastolic blood pressure. In our study, we found a NRs prevalence of 20 and 15% for decreased systolic blood pressure in the H-IR and L-IR groups, respectively, as well as a more pronounced NRs prevalence of 30 and 45% for decreased diastolic blood pressure in the H-IR and L-IR groups, respectively  and L-IR (+14.7%) groups (Table LE results in the H-IR group and no NRs (0%) in the L-IR group , blood pressure (55–100%), metabolic (25–95%), and performance (0–35%) variables. These results are consistent with literature reports for blood pressure (59–60%) , no differences were observed in the NRs prevalence with regards to improved HOMA-IR scores, other anthropometric, cardiovascular, and muscle performance variables after 10 weeks of HIIT in sedentary adult women. This research demonstrates the protective effect of HIIT against cardiometabolic disease progression in a sedentary population.CA conceived and designed the project. CA and RR-C reviewed the literature studies and conducted data extraction. CA conducted data analyses. CA, RR-C, and MI were responsible for data interpretation. CA, RR-C, and MI drafted the manuscript, and RR-V, and MI revised it critically for intellectual contributions. CA and RR-C coordinate the study development. All authors reviewed and edited the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AS and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Experienced sprinters are specifically adapted to pre-planning an advanced motor program. Herein, sprinters are able to immediately accelerate their center of mass forward with a whole-body coordinated motion, following a steady state crouched position. We examined the effect of variable timing of reaction signals on multiple joint reaction times (RT) and whole-body RT for specialist sprinters. Twenty well-experienced male sprinters performed five start-dashes from a block start under five variable foreperiod (FP) length conditions , with trials randomly timed between a warning and an imperative tone. Participants’ sprinting motion and ground reaction forces of their four limbs during the block start were measured simultaneously. Whole-body RT was significantly shorter when FP length was longer; the values of whole-body RT were 117 ± 5 ms, 129 ± 5 ms, 125 ± 4 ms, 133 ± 6 ms, and 156 ± 8 ms in the 2.096, 1.938, 1.780, 1.622, and 1.465-s FP-length conditions, respectively. A repeated-measures analysis of variance found a significant joint-by-FP length interaction in joint-moment RT. These findings suggest that FP length affects coordinated motion in four limbs and whole-body RT. This information will be able to lead to new methods for start signals in sprint running events and advance our understanding of the association between FP length and dynamic coordinated motion. Experienced sprinters are specifically adapted to accelerating their center of mass immediately forward after being in a steady set crouched position . This blReaction time (RT), which is the duration from response signal onset to the first discernible change in focal force-generation activity, is influenced by the anticipation of the timing of the stimulus input e.g., . The RT RT in block start has already been researched in the past e.g., . In a coPrevious basic studies measuring surface EMG activity have revealed that the optimal FP length (with the shortest EMG latency) differs in accordance with the muscle . For insThe purpose of this study was to clarify the effect of variable FP length in the block start signal on subsequent whole-body coordinated pattern and whole-body RT for experienced sprinters. This information may provide evidence-based advice for sprinters, coaches, and organizations building a common athletic regulation. Our hypothesis was that longer FP length in the block start signal based on recent athletic races would affect whole-body coordinated pattern and shorter whole-body RT.n = 2), Summer Universiade (n = 2), and similar-level international games (n = 3). All experiments were conducted after obtaining approval from the Research Ethics Committee involving Living Human Participants in Biwako Kusatsu campus, Ritsumeikan University; this study was performed in accordance with the ethical guidelines of the Declaration of Helsinki. All participants gave written informed consent before participating in the study.Twenty male sprinters participated in this sprint start experiment . Seven participants participated in the World Championships . Based on the distribution of the measured FP length, five FP lengths for the experiment were determined by the mean and SD: mean – 2 SD (1.465 s), mean – 1 SD (1.622 s), mean (1.780 s), mean + 1 SD (1.938 s) and mean + 2 SD (2.096 s) . The warning and imperative tones were generated using Matlab  and a trumpet horn  placed beside the participants approximately 30 cm from the ear. The warning and imperative tones were set with intensities of 70 and 120 dB, respectively, which were used in a previous study on startle response in a block start . Onsets of the starter’s set and gun signals were visually determined by sound wave information. ck start . These iThe experimental trial involved five 5-m dashes from a block start under a variable FP-length condition with the trial randomly timed between the warning and imperative tones. Participants were instructed to start as quickly and strongly as possible without anticipating the go signal. The rest duration between trials was 3 min. In order to avoid participants from accurately anticipating the go signal timing, a catch trial in which the go signal was not delivered was used . The catFigure 2 shows the detailed marker set for the whole body and starting blocks. The three-dimensional locations of the markers were recorded using a 16-camera motion capture system  sampling at 250 Hz. The starting blocks were adjusted onto separate force plates  during the block start were captured simultaneously. A total of 60 retro-reflective markers sized 12 mm were attached to the whole body and the starting blocks. e plates . GRFs geA 15-segment rigid body model was created, with the head, trunk, pelvis, upper arms, forearms, hands, thighs, shanks, and feet. Segmental anthropometric data was used from Figure 4A). Because the variability of time-series data of sum of GRFs or joint moment in the baseline zone was different based on individual differences and joints, a fixed SD was inappropriate to determine the onset instant of the pushing blocks or floor. Therefore, the SDs were adjusted in accordance with each trial; temporal RT was first determined by increasing the SD sampled at 0.1 ms, and the actual RT was determined if the difference between the temporal RT and the next temporal RT was over 10 ms . When GRF/joint moment data increased from the baseline (following a sufficient decrease), the GRF/moment threshold value was determined during the decrease. If the temporal RT was not clearly appropriate for the actual RT  using the detection method, the actual RT was finally checked by visually locating and manually adjusting the onset mark to the point at which the activity first increased/decreased.The RT generated by the sum of the GRFs of the upper and lower limbs (whole-body RT) and the RT generated by the moment in each joint (joint RT) were calculated using a threshold of the mean value ± the adjusted SD, which differed based on each participant and each joint, in the baseline zone .Data are presented as mean ± standard error (SE). All datasets were initially checked for normality and homogeneity of variance. To assess changes in whole-body RT and changes in joint RT within the same joint across the five FP-length conditions, a repeated-measures analysis of variance (ANOVA) was conducted. With regard to joint RT, a two-way repeated-measures ANOVA was conducted with FP length and joint as factors. The FP length × joint interaction was to clarify whether changes in the joint RT in the five conditions were different based on the joints and to assess the multi-joint coordination pattern in each condition. Bonferroni’s test was used in all Figure 5 shows a typical example of whole-body RT and RT of joint moments during the stance phase in the five conditions (participant #11). For the LL condition, after the gun signal, the front-arm shoulder, already generating a flexion moment at the set position, was the first to react (joint RT: 61 ms), reducing the flexion moment. A similar time-moment curve was observed in the rear-arm shoulder; however, joint RT was delayed compared to the front-arm shoulder (joint RT: 123 ms). Next, the rear-leg knee (joint RT: 76 ms), ankle (joint RT: 109 ms), and hip (joint RT: 111 ms) reacted to the gun signal, generating an extension moment during the stance phase after nearly 0 Nm at the set position. In all joints in both legs, the front-leg knee was the last to react to the gun signal, generating an extension moment during the stance phase (joint RT: 131 ms). Similar extension force-generation patterns within the three joints of the front-leg were observed during the stance phase. The elbow and wrist joint flexion moments were reduced at the moment of take-off, already generating flexion moments at the set position. However, the values were closer to 0 Nm compared to the other joint moments. Whole-body RT for participant #11 was 108 ms in the LL condition. In the other four conditions, similar time-moment curves were observed for participant #11; however, the joint RTs were delayed compared to the LL condition.Table 1). In the SS, S, N, L, and LL conditions, whole-body RT were 156 ± 8 ms, 133 ± 6 ms, 125 ± 4 ms, 129 ± 5 ms, and 117 ± 5 ms, respectively. Whole-body RT in the SS condition was significantly longer compared to that in the N, L, and LL conditions; the differences were 31 ± 7 ms, 26 ± 8 ms, and 39 ± 7 ms, respectively.As shown by the data for mean ± SEM across all participants, whole-body RT was significantly shorter when the FP length was longer  = 2.234, P < 0.05]. In the front-leg hip and ankle; rear-leg hip, knee and ankle; front-arm shoulder and elbow; and rear-arm shoulder, elbow and wrist joints, the joint RT was significantly shorter in the longer FP-length conditions (Table 2). In contrast, there were no significant changes in joint RT in the front-leg knee and front-arm wrist among the five FP-length conditions.A two-way repeated-measures ANOVA found that there was a significant FP length × joint interaction on joint RT [Table 3). In contrast, in the S condition, a positive relationship was observed between only the front-arm shoulder joint RT and, in the N, L, and LL conditions, no significant relationships were observed between whole-body RT and joint RT. Stepwise multiple regression analysis clarified that the rear-arm wrist joint RT was closely to whole-body RT in the SS condition, and the front-arm shoulder joint RT was closely related to whole-body RT in the S condition (Table 4). In contrast, in the N, L, and LL conditions, no joint RT was found to be closely related to whole-body RT.In the SS condition, positive relationships of whole-body RT were significantly observed between front-leg hip and knee joint RT, rear-leg knee and ankle joint RT, front-arm wrist RT and rear-arm shoulder and wrist joint RT  was longer than whole-body RT (156 ± 8 ms) in the SS condition. Similarly, in the S condition, the front-arm shoulder joint RT (143 ± 8 ms), which was a closely related joint RT to whole-body RT, was longer than whole-body RT (133 ± 6 ms). Therefore, even though cross-sectional relationships were observed between whole-body RT and closely related joint RT under shorter gunfire timings, no main contributor for whole-body RT is probably involved for many sprinters. In the N, L, and LL conditions, whole-body RT was significantly related to any joint RT to begin with. These findings suggest that multi-joint coordination of dynamic beginning motion is changed by the unanticipated timing of loud auditory stimuli and the main joint determinant for whole-body RT is based on the individuality of sprinters.At a set position, relative to the whole-body center of mass, the contacting hands anteriorly and feet posteriorly leads sprinters to maintain stability. The position of shoulders being forwarded at set position generating the flexion moment, arms allows crutch . After tShoulder joints, which are located in the most proximal position to the central nerve system, reacted first to the imperative signal more than other joints in the N, L, and LL conditions. Interestingly, in the LL conditions, the shoulder joint initially reacted within 100 ms. This suggests that shoulder joints of both arms more sensitively contribute to whole-body RT rather than the other joints in the longer FP-length conditions. As shown for participant #11, whose front-arm shoulder joint RT was 61 ms, it can be considered that the shoulder joints of some sprinters react very quickly to a long gunfire timing. Thus, there is a possibility that participant #11 may have anticipated the go signal. Fortunately, whole-body RT of participant #11 was slightly over 100 ms; however, this results may indicate that, for some sprinters exposed to a longer gunfire timing, this initial shoulder motion might lead to a false start motion in competitive races.The difference in whole-body RT lengths between the shortest- and longest-RT length conditions, the SS and LL conditions, was 39 ± 7 ms, corresponding to the RT difference in previous studies with similar FP lengths . This suThis study has three limitations. First, the auditory data of the ‘set’ and gunfire in the athletic event were captured using that in television broadcasts, and thus we could not identify how the starter’s voice and gunfire were recorded. Therefore, the FP length that we measured likely involve some error compared to that which sprinters actually heard in the athletic event. Second, the number of trials was limited to one trial in each condition. We could not clarify RT variability in each individual sprinter; therefore, the RT of some participants may involve measurement error. However, according to a previous study , the numThis study demonstrated that long FP length, as determined by FP length in athletic games over the last 5 years, affects the subsequent short joint moment and shorter whole-body RT in block start. This information could lead to new methods for start signals in sprint running events and could explain the fundamental effect of anticipatory RT in whole-body reaction motion in humans.Conceived and designed the experiments: MO and TK. Performed experiments: MO and TK. Analyzed data: MO. Interpreted results of research: MO, TK, and TI. Drafted manuscript and prepared tables/figures: MO and TK. Edited, critically revised paper and approved final version of manuscript: MO, TK, and TI.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study investigates whether the diverse responses mediated by IL-17 in vivo also lead to enhanced BMP-2-induced bone formation. Since IL-17 is known to induce osteoclastogenesis, we studied the interactions between IL-17 and BMP-2 in ceramic scaffolds either or not carrying a coating with the bisphosphonate zoledronic acid (ZOL). Histological evaluation revealed that IL-17 alone did not induce any osteoclasts at day 10. On the other hand, BMP-2 clearly stimulated early tissue ingrowth and osteoclastogenesis. Both of these processes were blocked in presence of ZOL. IL-17 signaling restored early vascularized connective tissue formation and osteoclastogenesis induced by BMP-2 in ZOL-coated scaffolds. After 12 weeks, the bone volume induced by co-delivery of BMP-2 and IL-17 was doubled as compared to that induced by BMP-2 alone. We conclude that IL-17 has osteo-stimulatory effects through a synergy with bone-inductive BMP-2. Although local and single application of IL-17 does not mediate osteoclast formation, it could promote other processes involved in bone formation such as connective tissue ingrowth. The use of IL-17 may contribute to the development of improved bone graft substitutes.Interleukin 17 (IL-17) stimulates the osteogenic differentiation of progenitor cells  An effective bone substitute consists of an osteoconductive matrix incorporated with biological factors that maximize the osteogenic response2. Bone morphogenetic protein 2 (BMP-2) is regarded as the factor with the highest potential for bone induction, but its current use is considered to be inefficient and unpredictable3. The clinical BMP-2 dose (1-2 mg/cc carrier)4 far exceeds the native concentration of BMPs, i.e. in the order of ng/cc bone5. Serious side effects have been reported for BMP-2, such as ectopic bone formation, osteolysis, and even urogenital or neurological complications6. These side effects are likely related to its supraphysiological dose7, however attempts to lower the clinical dose also resulted in significantly reduced bone augmentation8. There is evidence that sequential or simultaneous delivery of BMP-2 with other pro-osteogenic stimuli may increase the responsiveness of cells to BMP-2. For instance, a biological effect of BMP-2 can already be seen for a concentration as low as 25 ng/ml following co-delivery with multipotent mesenchymal stromal cells (MSCs)9. Likewise, pro-inflammatory cytokines are thought to modulate bone regeneration in part through their interaction with BMP-2, suggesting that their co-delivery could potentiate the osteoinduction provided by BMP-211.There is a need for bone substitutes that can decrease the reliance on autologous bone grafts for repair of bone defects12. In bone research, IL-17 has been implicated in the progression of inflammation-mediated bone loss14. On the other hand, IL-17 may also show pro-osteogenic effects. For example, IL-17 promotes bone healing and is a mediator of pathological bone formation in conditions such as spondyloarthritis18.This study investigates a potential role of the pro-inflammatory cytokine interleukin 17 (IL-17) as a modulator of BMP-2-induced new bone formation. IL-17 is mainly produced by T lymphocytes and neutrophils, and is an important mediator of the inflammatory response in conditions such as trauma, infection and autoimmunity19. Although this suggests that IL-17 exerts a negative effect on the osteogenic response, its influence on bone regeneration could be heavily dependent on the context of IL-17 signaling. First, IL-17 alone is not osteoinductive in MSC cultures, yet it dramatically increases MSC matrix mineralization mediated by BMP-218. The investigation of possible synergistic actions between IL-17 and BMP-2 on in vivo osteogenesis is therefore of interest. Second, IL-17 signaling could be a double-edged sword in bone formation due to its stimulatory effects on osteoclast differentiation21. While a balanced osteoclast activity is normally coupled to osteoblast formation23, exaggerated osteoclastogenesis results in bone resorption and has a negative impact on new bone formation24. Therefore it is of relevance to determine if suppression of osteoclasts could skew IL-17-mediated effects towards enhanced bone formation. In the context of immunomodulatory strategies, the use of bisphosphonates such as zoledronic acid (ZOL) may be feasible, since they are widely used in clinical practice and have shown to enhance bone mass also when applied under inflammatory conditions25. The effect of IL-17 on osteoblastogenesis is studied in an ectopic location, where the formation of bone tissue relies completely on the recruitment and differentiation of MSCs towards the osteogenic lineage26. It has been shown that MSCs recruited from either bone marrow, circulation, or local vascularized tissue will home to bone scaffolds following ectopic implantation28. This methodology allows for better study of osteoinductive pathways compared to bone defect models, where native bone-inductive growth factors and cells support healing partially through an osteoconductive mechanism26. In addition to MSCs, macrophages are considered an important cell type contributing to new bone formation through local immune modulation, growth factor production, and/or providing osteoclastogenesis30.The mechanisms responsible for the pro-osteogenic effects of IL-17 are largely unknown. To our knowledge, only a single study has investigated the effect of exogenous IL-17 delivery on bone regeneration. This study showed attenuated calvarial defect healing when IL-17 was added to ceramic bone graftsin vitro, it was hypothesized that IL-17 could potentiate new bone formation induced by BMP-2. Effects of IL-17 were studied with respect to early tissue response and subsequent bone formation. To assess the role of osteoclasts in IL-17-mediated responses, scaffolds were used that were either or not coated with ZOL.In the present study, we investigate how IL-17 affects ectopic bone formation in ceramic scaffolds in rabbits. Following the observation that IL-17 acts in a synergistic manner with osteoinductive factors 31. The mechanical and biological properties of this ceramic (i.e. BCP 1150) have been described in detail elsewhere33. The blocks were autoclaved at 121 °C and dried at 60 °C before in vivo use. The relatively large scaffold size of 10 × 10 × 10 mm is still suited for subcutaneous implantations, while allowing stimulatory and inhibitory effects within a range of early bone formation processes. Considering the diffusion limit for oxygen of 100–200 μm34, the tissue ingrowth was expected to slowly occur from the edge of the implants towards the center.Porous biphasic calcium phosphate (BCP) blocks were used with a total porosity of 75 ± 5%. The material consisted of 20 ± 5% β-tricalcium phosphate (TCP) and 80 ± 5% hydroxyapatite by weightet al.35, who demonstrated increased bone formation in ectopic β-TCP scaffolds due to ZOL. The current IL-17 concentration range (1.3–133 ng/ml) was based on our own in vitro study18, in which we showed that IL-17 most strongly stimulated osteoblastogenesis at 50 ng/ml within a tested 5–500 ng/ml concentration range. We previously identified a BMP-2 concentration which resulted in a limited bone area% of 5% after 12 weeks in the same rabbit model10. A comparable suboptimal BMP-2 dose was used in the current study to discriminate the positive or negative effects of the mediators of interest.The concentrations of ZOL, BMP-2 and IL-17 in the BCP scaffolds were based on previous reports. The ZOL concentration was selected according to a study by Ichikawa 3 is the available space in the scaffolds. Control constructs received 110 μl MilliQ. The samples were air dried for 48 h at room temperature. Non-precoated and ZOL-precoated scaffolds were loaded with rhIL-17 , alone or in combination with rhBMP-2 , according to Table To prepare ZOL -coated scaffolds, 110 μl of a 228 μg/ml stock solution in MilliQ was pipetted onto the BCP blocks. This volume was completely absorbed by the scaffold. This resulted in an end concentration of 33.4 μg/ml, considering that 0.75 cm36. A mobile phase mixture was used consisting of methanol:water (10:90), with 6 mM tetrabutylammonium hydrogen as an ion pair for ZOL at pH 2.6. Samples were injected into the HPLC system  coupled with LiChrospher® C-18 column . The absorption was measured at 208 nm  at an elution speed of 0.8 ml/min. Results were analyzed with Waters Empower 3 Pro software.Non-coated and ZOL-coated scaffolds were incubated in 700 μl MilliQ in eppendorf tubes at 37 °C. At different time points, the scaffolds were removed and the supernatant was stored at −20 °C. Separate tubes were made for each time point in duplicate. The ZOL concentration in the supernatant was measured by High Performance Liquid Chromatography (HPLC) as described previouslyAnimal experiments were performed after approval of the local Ethics Committee for Animal Experimentation . All procedures were carried out in accordance with the approved guidelines, and all efforts were made to minimize the number of animals used and their suffering. A total of twelve male New Zealand White rabbits  were used, and were housed at the Central Laboratory Animal Research Facility, Utrecht University. One rabbit from the 10-day study died the day after surgery and the cause of death could not be identified by necropsy. The implants from this rabbit were retrieved within an hour after its death for histological analyses (24 h constructs). The missing rabbit was replaced to match the group sizes. All other animals recovered well from the surgery and implanted samples were all retrieved. Three rabbits were euthanized after 10 days to study the early tissue response. Eight rabbits were euthanized after 12 weeks to study bone formation  and glycopyrrolate  pre-operatively, and medetomidine  peri-operatively. Anesthesia was reversed with atipamezole hydrochloride . Antibiotic prophylaxis  was given once peri-operatively. Animals received pain relief pre-operatively, and post-operatively every 12 hours for 2 days with buprenorphine .The rabbits received ketamine  formaldehyde. For the 12-week study, a third of each sample was removed and decalcified in 0.3 M EDTA for paraffin embedding. The remaining material was embedded in methyl methacrylate. The constructs from the 10-day study were entirely decalcified and embedded in paraffin.Hematoxylin and eosin (H&E) staining was performed to evaluate general tissue morphology. To demonstrate connective tissue formation by Goldner’s trichrome staining, sections were deparaffinized and incubated with Weigert’s hematoxylin for 5 min, followed by Goldner’s solution  for 45 s, 2% (w/v) Orange G for 7 min and 0.15% (w/v) Light green for 7 min. Differentiation was done with 1% (v/v) acetic acid for 5 s after each staining. Samples were dehydrated and mounted in Depex. Sections were scored by two blinded observers for presence of connective tissue in either 0, 25, 50, 75 or 100% of the available space. Data are presented as the average of the two scores.in vitro resorptive activity37. The number of osteoclasts was counted in one cross-section from each construct and averaged per group.For detection of osteoclasts, staining for tartrate-resistant acid phosphatase (TRAP) activity was performed. Samples were incubated with 0.2 M acetate buffer-tartaric acid for 20 min at 37 °C. Naphtol AS-MX phosphate  and Fast red TR salt  were added for another 2 h. Sections were counterstained with Mayer’s hematoxylin. Osteoclasts were defined as multinucleated TRAP-positive cells lining the surface of bone or scaffold. An absolute osteoclast count was considered to be a reliable measure of osteoclastogenesis since it correlates with 10. The number of positive cells was counted in one cross-section from each construct and averaged per group.Stainings for calprotectin-expressing phagocytes  or T lymphocytes  were performed as described previouslyTo stain blood vessels, samples were incubated with anti-CD31  for 1 h at room temperature following heat-induced antigen retrieval with EDTA . Blocking and staining was performed with a kit, according to the manufacturer’s protocol .Using the same kit as the CD31 staining, cytoplasmic staining for connective tissue macrophages  was performed with overnight incubation of the primary antibody at 4 °C. Sections were counterstained with Mayer’s hematoxylin.39. Bone histomorphometry on MMA sections was therefore the best representative of the bone volume present in the scaffolds. Two MMA midsections were cut with a saw microtome  at 35 μm thicknesses and stained with basic fuchsin/methylene blue . The animals received each of the conditions listed in Table The datasets generated during the current study are available from the corresponding author on reasonable request.In vitro release measurements showed that the scaffolds retained approximately half of the loaded ZOL after an initial burst release, without any additional release at later time points  did not contain any bone. The groups that contained BMP-2 demonstrated the presence of dense lamellar bone and adipose tissue Fig. . BMP-2 wNo TRAP-positive cells were observed in empty scaffolds or scaffolds loaded with 100 ng IL-17 Fig. . Numerou18, IL-17 significantly enhanced BMP-2-induced new bone formation. We furthermore showed that IL-17 does not induce early osteoclastogenesis, but likely supports other processes involved in bone formation including vascularized connective tissue ingrowth.The objective of this study was to explore the possible role of IL-17 in the early and/or late osteogenic response. BCP scaffolds with a suboptimal amount of BMP-2, either or not combined with IL-17, were investigated in subcutaneous pockets in rabbits. In this location, IL-17-mediated effects can be detected on osteoinductive pathways leading to bone formation. In line with its promotive effect on MSC osteogenic differentiationet al., who found an inhibitory effect of IL-17 on calvarial bone defect healing in rats. They, and others, supported this in vivo finding by showing decreased osteogenic marker expression in calvarial osteoblasts in vitro42. As an explanation, the effects of IL-17 on bone formation could be dependent on the cells that are targeted in that specific model. We found a stimulatory effect of IL-17 on ectopic new bone formation, i.e. a process that relies on the migration and differentiation of inducible cells28. In agreement, IL-17 potently enhances further osteogenic differentiation of undifferentiated MSCs and other immature cells44. Thus, IL-17 may have different effects on bone cells depending on their differentiation stage. Furthermore, the IL-17 receptor expression levels may vary in bone cells isolated from various sources, which could explain variable efficacy of IL-17 depending on the target tissue43. In the current study, only IL-17A was investigated, commonly referred to as IL-1745. Other IL-17 family members may also be interesting to study, since we already confirmed a pro-osteogenic role for IL-17F in vitro18.In the current study, the combined delivery of BMP-2 and IL-17 led to a twofold greater bone volume as compared to BMP-2 alone. Our observation that IL-17 enhances bone formation contradicts the study by Kim 11. IL-17 was effective in the ng/ml range, resembling its expression levels during inflammation46. Furthermore, an in vivo burst release of IL-17 was expected, which mimics its short-lived action in the bone healing response15. The timely delivery of a physiological amount of IL-17 could be a potentially feasible method, as it circumvents the destructive effects of chronic IL-17 signaling45. Consequently, the co-delivery approach should be explored as a strategy to improve the efficacy of BMP-2. This is of great importance, since the high costs and side effects of BMP-2 are likely related to its inefficient use3.The observed synergistic interaction between IL-17 and suboptimal dose BMP-2 in terms of bone formation may recapitulate the natural crosstalk between pro-inflammatory and osteogenic signaling pathways during bone regeneration47. It is therefore crucial to evaluate whether IL-17 can enhance the bone mass and mechanical stability in functional models such as spinal fusion or critical-size defects50. It is furthermore of interest to study how IL-17 modulates the osteoconductive and/or osteoinductive properties of ceramics with a faster resorption rate, including certain TCP formulations, since they usually promote better healing in large bone defect models than the currently studied BCP33. As part of these studies, fluorochrome labeling could provide valuable insight in the location and dynamics of new bone deposition relative to the existing bone51.To further establish the potential of IL-17, it is of relevance to determine if its pro-osteogenic effects can also augment the healing of bone defects. As such, ectopic bone models neglect parameters that will ultimately direct the bone healing response in clinical scenario’s, such as presence of bone-stimulating cytokines, bone-forming cells, or bone-mechanotransduction41, we found that the presence of high numbers of osteoclasts precedes new bone formation in the osteogenic constructs. To test the hypothesis that IL-17-mediated effects on osteoclastogenesis hampers its pro-osteogenic effects24, IL-17 in combination with the osteoclast-inhibitor ZOL was studied. To this end, a local ZOL dose (25 μg/scaffold) was applied that was considered clinically-relevant, as the systemic dose used to treat bone loss is approximately 60 μg/kg52. Consequently, the ZOL coating was found to be very effective in blocking osteoclast presence in the BMP-2-stimulated condition. In contradiction to the hypothesis, there was no obvious stimulatory effect of IL-17, alone or combined with BMP-2, on the number of osteoclasts. Furthermore, changes in early osteoclast activity did not necessarily affect the osteogenic response. Whereas osteoclasts play a prominent role in cell- or material-induced ectopic bone formation29, we found similar BMP-2-mediated bone formation ZOL-coated scaffolds as compared to non-coated scaffolds. In addition, others have shown that appropriate timing and/or administration route of ZOL treatment can lead to more favorable bone formation by preventing osteolysis caused by supraphysiological dose BMP-255. We conclude that the pro-osteogenic effects of IL-17 cannot be convincingly attributed to changes in osteoclastogenesis.In line with other observations56. The vascularized connective tissue ingrowth seen at day 10 in constructs with BMP-2 was strongly impaired by ZOL. Several mechanisms could be responsible for this inhibitory effect, depending on the ZOL concentration and timing. For instance, ZOL inhibits blood vessel formation induced by vascular endothelial growth factor (VEGF) and has pro-apoptotic effects on endothelial cells. Equally important, ZOL has negative influences on the survival and function of bone (precursor) cells depending on the applied concentration58. The negative impact of ZOL on BMP-2-induced vascularization and connective tissue formation were counteracted by IL-17. In line with these findings, IL-17 contributes to wound healing through the recruitment of other inflammatory cells and the modulation of angiogenesis59. MSCs could be a particularly important target, showing changes in migration, proliferation/differentiation and cytokine expression in response to IL-1762. Hence, the identification of the different targets of IL-17 in the osteogenic response is an important focus of further investigation.Alternatively, we provide evidence that other early processes critical for bone formation may be positively influenced by IL-17. As part of the TGF-β superfamily, BMP-2 plays a role in tissue repair by stimulating cell recruitment, MSC proliferation/differentiation and extracellular matrix production15. In line, we found a synergistic interaction between IL-17 and BMP-2 using a burst release approach. It remains of interest to establish whether timing is important with regard to the osteogenic response enhanced by IL-17. It is generally thought that prolonged pro-inflammatory cytokine signaling impairs bone formation through several mechanisms, including decreased extracellular matrix production, delayed angiogenesis and/or uncontrolled cell death64. When studying prolonged IL-17 signaling using a long-term release system65, several systemic parameters are increasingly important to take into account. First, possible detrimental effects of chronic IL-17 signaling on systemic inflammation or generalized bone loss should be closely evaluated66. Second, detection of circulating IL-17 levels could exclude possible interference with distant sites. As the studied mediators all have a relatively short systemic half-life (minutes-hours)69, it was estimated that the incorporated factors had no or very limited effects on other ectopic sites.The induction of IL-17 is limited to the first few days after bone injury29. In addition, cell isolation and detection of specific cell populations by fluorescence activated cell sorting could for example provide valuable information regarding the osteoblast/osteoclast ratio’s in the scaffolds15.Several recommendations can be made for future work. Initially, relatively few experimental animals were used to explore the effects of IL-17 with respect to early tissue response. Accordingly, the reported direction and effect size induced by IL-17 and/or ZOL on the observed histological changes will allow for more precise sample size calculations in future quantitative studies. Furthermore, due to incomplete connective ingrowth in the day-10 samples and tissue loss during processing, the center of the scaffolds could not be evaluated for CD31 and RAM11 expression. For the same reason, reliable immunodetection of osteoblasts was not possible at early time points. Scaffold homogenization and measurement of bone markers at the mRNA level could overcome this issue in the futureIn the present study, we identify a novel role of IL-17 in potentiating bone substitutes. It was observed that IL-17 acts in a synergistic manner with BMP-2 to enhance new bone formation, but the mechanism by which IL-17 mediates this remains elusive. In addition to its stimulatory effect on MSC osteogenic differentiation, we showed that IL-17 may promote soft tissue ingrowth. On the other hand, IL-17 does not promote osteoclastogenesis. Together, these findings suggest that the induction of IL-17 signaling is a feasible strategy to promote bone regeneration.


Lead is harmful for human health and animals. Proanthocyanidins (PCs), a natural antioxidant, possess a broad spectrum of pharmacological and medicinal properties. However, its protective effects against lead-induced liver damage have not been clarified. This study was aimed to evaluate the protective effect of PCs on the hepatotoxicity of male Kunming mice induced by chronic lead exposure. A total of 70 healthy male Kunming mice were averagely divided into four groups: control group, i.e., the group exposed to lead, the group treated with PCs, and the group co-treated with lead and PCs. The mice exposed to lead were given water containing 0.2% lead acetate. Mice treated in the PCs and PCs lead co-treated groups were given PC (100 mg/kg) in 0.9% saline by oral gavage. Lead exposure caused a significant elevation in the liver function parameters, lead level, lipid peroxidation, and inhibition of antioxidant enzyme activities. The induction of oxidative stress and histological alterations in the liver were minimized by co-treatment with PCs. Meanwhile, the number of Transferase-Mediated Deoxyuridine Triphosphate-Biotin Nick End Labeling (TUNEL)-positive cells was significantly reduced in the PCs/lead co-treated group compared to the lead group. In addition, the lead group showed an increase in the expression level of Bax, while the expression of Bcl-2 was decreased. Furthermore, the lead group showed an increase in the expression level of endoplasmic reticulum (ER) stress-related genes and protein (GRP78 and CHOP). Co-treated with PCs significantly reversed these expressions in the liver. PCs were, therefore, demonstrated to have protective, antioxidant, and anti-ER stress and anti-apoptotic activities in liver damage caused by chronic lead exposure in the Kunming mouse. This may be due to the ability of PCs to enhance the ability of liver tissue to protect against oxidative stress via the Nrf2/ARE signaling pathway, resulting in decreasing ER stress and apoptosis of liver tissue. Lead is a non-essential toxic heavy metal which has been a persistent public health problem in many countries around the world. Lead is an environmental toxicant which causes a broad range of adverse effects in both humans and animals . In the As lead is a multi-target toxicant, it exerts a toxic manifestation by oxidative free radicals that mediate the disruption of the delicate pro- and anti-oxidant balance existing in mammalian cells, and a therapeutic strategy to elevate the antioxidant defenses of the body may be of assistance in protecting from lead toxicity. Recently, several anti-oxidative approaches have been proposed to alleviate the symptoms of lead damage ,13,14. HProanthocyanidins (PCs), also named condensed tannins, are oligomers and polymers of flavan-3-ols, contain various amounts of catechin and epicatechin , widely However, to our knowledge, there are no similar studies available on the protective effects of PC on lead-induced hepatotoxicity in mice. If PCs have the ability to protect the liver, a diet comprising PCs would serve as useful clinical medicine against lead toxicity. Therefore, this study was aimed to elucidate whether PCs can prevent oxidative stress-induced hepatotoxicity and its possible mechanism.Male Kunming mice (15 ± 0.5 g and three weeks old) were purchased from the China Medical University . Mice received food and water randomly. All stress factors were reduced to a minimum. The experiments have been allowed by the ethics committee for laboratory animal care  for the use of Shenyang Agricultural University, China (Permit No. SYXK<Liao>2011-0001).l-epicatechin (4.54%), and were diluted in distilled water before use. The mice anti-Nrf2 and anti-γ-GCS antibodies were acquired from Santa Cruz biotechnology . The citatory number of the anti-Nrf2 antibody was sc-722, the anti-γ-GCS antibody was sc-22755, and the anti-GRP78 antibody was sc-1050. Anti-HO-1 and β-actin antibodies were obtained from Sangon Biotech ; anti-CHOP antibody was purchased from Beyotime Biotech ; these antibodies were all polyclonal antibodies. We also purchased the antibodies conjugated with the secondary goat anti-mouse and goat anti-rabbit horseradish peroxides (HRP) from Beijing Solarbio Science and Technology Co., Ltd. . SYBR green RT-PCR kit from Takara  and 4′,6-diamidino-2-phenylindole (DAPI) from Sigma Aldrich  were also employed. The primers for Nrf2, HO-1, γ-GCS, GRP78, CHOP, and β-actin were synthesized and purified by Sangon Biotech . Moreover, the preservation solution of RNA samples and the kits for total animal RNA extraction were obtained from Sangon Biotech Co., Ltd. . The kits for Revert Aid First Strand cDNA synthesis were purchased from MBI Fermentas ; kits for the testing of glutathione peroxidase (GSH-Px), reduced glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were obtained from Nanjing Jiancheng Bioengineering Institute .Standard lead was purchased from the National Standard Material Research Center . Lead acetate was purchased from Shanghai Biochemical Technology Co., Ltd. . PCs extracted from grape seeds (purity > 95%) were obtained from Zelang Medical Technology Company . The extract contained oligomeric proanthocyanidins (88.36%), catechin (6.68%), n = 7), mice were given 0.9% physiological saline at 1 mL/kg b.w.; lead acetate group (n = 21), mice were given 0.2% lead acetate in their drinking water for six weeks along with 0.9% saline at 1 mL/kg b.w; PC group (n = 21), mice were given PC in 0.9% physiological saline at 100 mg/kg b.w. for six weeks by oral gavage for six days every week and stopped for one day; lead co-treatment with PC group (n = 21), mice were given 0.2% lead acetate in drinking water with PC at a dose of 100 mg/kg b.w. every day for six weeks.Mice were allowed to acclimatize for one week prior to commencing experiments. Mice were given standard granulated food and drinking water and were divided randomly into four groups, as follows: control group (3 to dissolve the residue. Lead content was determined by a graphite furnace atomic absorption spectrophotometer.Whole blood (0.5 mL) and liver tissue (30 mg) samples were taken from mice and then mixed with 0.5 mol/L HNOFrom the serum samples, commercially available enzyme linked immunosorbent assay (ELISA) kits were used to test the activities of ALP, ALT, and AST. Experimental procedures were carried out according to the manufacturer’s instructions . In brief, the samples were transferred into a new 96-well plate containing substrates or buffer solution. After incubation at 37 °C, the plate was incubated for an additional time after adding a color developing agent and the absorbance at 510 or 520 nm was measured.The liver tissue was weighed and diluted to 10% liver homogenate and centrifuged for 15 min at 3000 rpm. The supernatant was removed and used for analysis. The contents of MDA and reduced GSH, and the activities of GSH-Px and SOD were determined according to the instructions of the manufacturer for the specific kit (Nanjing Jiancheng Institute of Biotechnology). The concentration of MDA was assayed by monitoring thiobarbituric acid reactive substance formation. According to the instructions of the reduced glutathione assay kit (Nanjing Jiancheng Institute of Biotechnology), the concentration of reduced GSH was detected by the Dinitrothiocyano benzene (DNTB) rate colorimetric method.2O, 1 μL fluorescein isothiocyanate (FITC)-labelled dUTP, and 1 μL terminal deoxynucleotidyl transferase (TdT) enzyme solution was mixed and added to the surface of one slide and incubated at 37 °C, for 1 h, in the dark. The sample was then strained by DAPI for 8 min. The labelled slices were washed and photographed under a fluorescence microscope. The nucleus of any apoptotic cells were brown, and the other nuclei were blue.Mice liver paraffin slices were prepared for TUNEL assay, which was performed by using a commercial kit in accordance with the manufacturer’s instructions. Briefly, the paraffin slices were fixed with dimethylbenzene for 15 min at the room temperature and washed with absolute ethyl alcohol twice, for three minutes each time, then washed with phosphate-buffered saline (PBS) for the third time, for five minutes. The fixed sections were incubated with 100 μL of 20 μg/mL proteinase K solution for 10 min. Subsequently, the 10 μL 5× reaction buffer, 38 μL ddHThe total protein and nuclear protein in 100 mg of the liver tissue of mice were extracted using Radio-Immunoprecipitation Assay (RIPA) lysis solution and a nuclear/cytoplasm protein extraction kit . The Bicinchoninic acid (BCA) protein assay kit  was used to determine the protein concentrations, and equivalent amounts of proteins (30 μg/lane) were loaded onto 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (SDS-PAGE). After the proteins were separated by SDS-PAGE, they were transferred to a Modified as polyvinylidene fluoride (PVDF) membrane. Antibodies then were added for rabbit anti-Nrf2 (1:1000), anti-HO-1 (1:1000), anti-γ-GCS (1:500), anti-Bcl-2 (1:1000), anti-Bax (1:1000), and β-actin (1:2000) polyclonal antibodies, and incubated overnight at 4 °C. On the second day, HRP-labeled goat anti rabbit IgG was added and incubated at room temperature for 2 h. An Electrochemical luminescence (ECL) chemical luminescence method was then used to detect the color reaction. Images were acquired and the results of the gray scale image were analyzed using a Bio-Rad gel imaging system. The relative expression levels of proteins in each group were represented by the ratio of the gray value of the target protein and the β-actin.® Premix Ex TaqTM, 6.8 μL of RNase-free water, and 0.4 μL ROX Reference Dye II (50×). The conditions of conducting the PCR reaction included: at the initial stage, denaturing at 95 °C for 5 min, and then denaturing at 95 °C for 15 s, annealing at 56 °C for 30 s, and extension at 72 °C for 30 s. The amount of the template was measured based on the standard curve of quantitative analysis. The primers of Nrf2, γ-GCS, Bcl-2, Bax, GRP78, and CHOP were designed by using Primer 5 software   at 260/280 nm (1.8–2.0). The 4 μg of total RNA from each sample was treated with DNAse. After DNAse treatment, 3 μg of DNA-free RNA in a total volume of 20 mL was then reverse transcribed into cDNA using MBI Ferments Prime Script RT reagent kit  according to the manufacturer’s instructions. The cDNA was used as the template for further quantitative RT-PCR analysis. An ABI 7500 real-time PCR system and the SYBR Green PCR Kit were used to conduct real-time PCR. Each sample had been measured in triplicate. For qRT-PCR reactions, 2 μL product of cDNA, 0.4 μL reverse primers, 0.4 μL forward, 10 μL 2× SYBRCA, USA) , and β-ap-value < 0.05 was considered as significant.All data were processed using SPSS 17 statistical software . The significance level of statistic differences between mean values was determined using one-way ANOVA, and a p < 0.05). The experimental model was successful as a system to determine the effects of lead in the presence and absence of PCs in mice. However, lead levels in the blood and liver tissue of mice co-treated with lead and PCs showed no significant difference compared with the animals treated only with lead (p > 0.05), indicating that PCs did not significantly improve the status of the lead in the blood and liver tissue of mice.As shown in p < 0.05). However, the activity of three enzymes in the group co-treated with PCs was significantly lower compared with those of the lead-treated group. These activities of these enzymes were not significantly altered in the PCs-only treated group compared with the control group. Co-treatment with PCs significantly reduced the activities of ALP, ALT, and AST (p < 0.05). The ALP, ALT, and AST levels in the lead group were higher than the control group.As shown in p < 0.05) and in comparison to PCs-co-treated animals. GSH was significantly increased (p < 0.05) in the group co-treated with lead and PCs compared with the animals only treated with lead. The levels of MDA were also significantly decreased (p < 0.05) in the co-treated group compared with the group treated only with lead. These results indicated that administration of PCs significantly improved the lipid peroxidation damage in the liver tissue in mice exposed to lead.As shown in The results of the liver histopathological aspect are shown in p < 0.05) (p < 0.05).Compared with the control group A, the TU < 0.05) C, which  < 0.05) D. Howevep < 0.05), whereas the expression of Bax mRNA was up-regulated in the lead group (p < 0.05). However, compared with the lead group, the significant up-regulation of Bcl-2 mRNA (p < 0.05) and down-regulation of Bax (p < 0.05) were shown in the group co-treated with lead and PCs. Consistent with real-time PCR, the Western blot findings also showed low expression of Bcl-2 protein in lead-treated mice (p < 0.05), while moderate to strong expression was observed for Bax (p < 0.05). Compared with the lead-treated mice, those co-treated with PCs exhibited improvement, represented by increased expression of anti-apoptotic Bcl-2 protein and moderate expression of the pro-apoptotic protein Bax in the liver of mice (p < 0.05).As we know, Bcl-2 has a role in cells that inhibits the signal which induces apoptosis, Bax has a role in promoting cell apoptosis, and in order to analyze whether PC protects against the toxicity caused by lead related to inhibiting cell apoptosis in the liver of mice, the expression of Bcl-2 and Bax associated with apoptosis was examined, As shown in ad group B , while these protein expressions were increased with significant difference in the PCs group (p < 0.05). Compared with the lead group, all of their expressions in the lead/PCs co-treated group were increased with significant difference (p < 0.05) , while these mRNA expressions were increased with significant difference in the PCs group. Compared with the lead group, the mRNA expressions of these genes in the lead/PCs co-treated group and in PCs group were increased with significant difference (p < 0.05).To analyze whether Nrf2 activation plays a role in PC protection against the toxicity caused by lead, the expression of Nrf2 and Nrf2-target proteins, HO-1 and γ-GCS, in the liver of mice were measured. As shown in  < 0.05) A–C. Meanp > 0.05), while these protein expressions were not significantly different in the PCs group. Compared with the lead group, all of the protein expressions in the lead/PCs co-treated group and PCs group were decreased with significant difference (p < 0.05) (Grp78 and Chop mRNA expression levels in the lead-treated group were increased with significant difference (p < 0.05), while these mRNA expressions were not significantly different in the PCs group. Compared with the lead group, the mRNA expression of these genes in lead/PCs co-treated group and in PCs group were decreased with significant difference (p < 0.05).To analyze whether the protective effect of PCs on liver damage was related to inhibiting cell ER stress caused by lead, we measured the expression of GRP78 and CHOP, which were the ER stress-typical related proteins. As shown in  < 0.05) A,B. MeanIn the present study, the choice of the doses of lead and lead exposure time was based on preliminary experiments by Wang et al. ,33, whicThe toxicity of the lead has been relatively well studied, particularly in the context of neuro- and nephrotoxicity. As the liver is the central organ in the processing and removal of harmful substances from the body, it is also highly susceptible to damage. In addition, because the liver, via the portal vein, is the first organ exposed to internally-absorbed Pb and is one of the major organs implicated in the storage, biotransformation, and elimination of Pb, the liver must be the target organ of lead and liver will be damaged by lead when lead is taken into the body. Moreover, the liver is the body’s largest detoxification organ, a lot of detoxification enzymes, such as cysteine proteases, will be affected by lead. Previous studies have shown that lead exposure can cause oxidative damage in the liver by inhibiting the activities of antioxidant enzymes, decreasing the concentration of non-enzymatic antioxidants and increasing the concentration of reactive oxygen species ,37,38. OPCs are highly efficient natural antioxidants, their antioxidant activity is 50 times higher than that of vitamin E and 20 times that of vitamin C, and their effects have been reported in a wide range of studies ,45,46,47Currently, Nrf2 is the key molecule which mediates the response of the endogenous antioxidant system. Activation of Nrf2 can promote the expression of antioxidant genes and induce synthesis of phase II detoxifying enzymes . StudiesIn our study, we found that the gene transcription level of Nrf2 and the expression protein level of Nrf2 were both increased in the lead group. This result indicated that the expression of Nrf2 could be induced by lead under chronic lead poisoning conditions. At the same time, the results indicated that under the chronic lead poisoning, in order to cope with the oxidative stress induced by lead, the cells opened the Nrf2/ARE signal pathway to compensate for the oxidation; however, the cells were still in a state of oxidative stress. When the mice were co-treated with PCs, the nuclear Nrf2 mRNA and protein expression were also increased with a significant difference compared with that in the control group and lead group. This result indicated that PCs could activate the expression of nuclear Nrf2 protein, indicating that the effect of PCs in antagonizing lead-induced oxidative stress in liver tissue was related to the Nrf2/ARE signaling pathway.Previous studies have suggested that a variety of cytokines and antioxidants could counteract the action of such chemicals causing up-regulation of HO-1 and γ-GCS expression ,55,56,57Apoptosis plays a key role in some metal-induced toxicity. Lead exposure can induce apoptosis of nerve cells , liver cPrevious studies showed that lead could induce endoplasmic reticulum (ER) stress responses in the nervous system ,63. Our Recent studies have provided evidence that PCs can be used in the treatment and prevention of diseases, such as atherosclerosis, gastric ulcers, cataracts, and diabetes. For the first time, our results suggested that the protective effect of PCs on lead-induced liver damage by inhibiting the ER stress and apoptosis is due, at least in part, to their anti-oxidant stress activity and their ability to modulate the Nrf2/ARE signaling pathway. These findings may be attributed to the manifold effects of PCs as functional foods in future applications.In conclusion, PCs could protect against damage resulting from ER stress and apoptosis induced by chronic lead exposure in the liver tissue of mice. The hepatoprotective effect of PCs might be that PCs activated the expression of HO-1and γ-GCS via the Nrf2/ARE pathway, resulting in decreasing ER stress and apoptosis of the liver tissue cells.


E)-2-hexenal, 1-hexanol, and (Z)-2-hexen-1-ol showed great accumulation in white-fleshed peach fruit.First purchases of fruit are mainly dependent on aspects of appearance such as color. However, repeat buys of fruit are determined by internal quality traits such as flavor-related volatiles. Differences in volatile profiles in white- and red-fleshed peach fruit are not well understood. In the present study, peach cultivars with white- and red-fleshed fruit were subjected to sensory analysis using electronic nose (e-nose) to evaluate overview volatile profiles. Approximately 97.3% of the total variation in peach color-volatiles was explained by the first principle component 1 (PC1) and PC2. After analyzing sensory differences between peach fruit samples, 50 volatile compounds were characterized based on GC-MS. Multivariate analysis such as partial least squares discriminant analysis (PLS-DA) was applied to identify volatile compounds that contribute to difference in white- and red-fleshed peach fruit cultivars. A total of 18 volatiles that could separate peach fruit cultivars with different colors in flesh during ripening were identified based on variable importance in projection (VIP) score. Fruity note latone γ-hexalactone had higher contents in red-fleshed cultivars, while grassy note C6 compounds such as hexanal, 2-hexenal, ( The peach is one of the most important fruit trees worldwide, and its fruit production was approximately 25 million tonnes in 2016 worldwide according to the Food and Agriculture Organization of the United Nations (FAO). China, Spain, and Italy produce approximately 70% of the peach crop in the world in 2016. The huge demand of peach fruit in human diet is due to its visual features, nutritional features, and unique flavour for consumption. Fruit color is the most important factor for consumers’ first-time purchases. The fruit flesh of most peach fruit is white or yellow in color. Meanwhile, there are a number of red or blood-fleshed peach cultivars grown in Asia and Europe ,4,5 but Approximately 100 volatiles have been identified in peach fruit . These cThe formation of red-fleshed peach is due to the activation of anthocyanin biosynthesis by transcription factors such as NAC and MYB ,15. HighTwo groups of peach fruit samples were used in the present study: white- and red-fleshed cultivars. Peach fruit samples were collected from the Melting Peach Institute  and an orchard at Xian, China. Fruit was harvested at commercial mature stage and stored at 20 °C for 3 days to allow postharvest ripening. There are three replicates of five fruit each in the present study. In brief, there were eight peach cultivars and all of the peaches were stored for 0 d and 3 d, resulting in 48 samples for further analysis. Fruit ripening related quality indices such as total soluble solids (TSS) and firmness were described in -1 with a final penetration depth of 10 mm and data are presented in Newton (N).Total soluble solids (TSS) were measured by slicing both ends of each of the five samples of fruit for each biological replicate. Two drops of juice from each side were then applied to an Atago PR101α digital hand-held refractometer. Fruit firmness was measured according to method described [2) (n-type semiconductor). Type T has the sensitive layer placed on a tube of aluminum, while the sensitive layer of type P is placed on a plain substrate. The LY2 sensors are metal oxide sensors based on chromium titanium oxide (Cr2 − xTixO3 + y) and on tungsten oxide (WO3).The volatiles produced by peach fruit were evaluated using e-nose FOX 4000  with three metal oxide sensor chambers equipped with 18 metabllic oxide sensors, according to the methods of our pevious study . There a−1. The measurement phase lasted for 120 s for each sample, and the clean phase was 240 s. The 0 s and 100 s of response values of sensors in the e-nose were used as the e-nose data of each sample for further data analysis.Frozen flesh tissues were ground into powder by grinding in liquid nitrogen and transferred to a centrifuge tube containing 5 mL saturated sodium chloride solution. After mixing, 2 mL of homogenate were transferred into a 10 mL crimp-top vial and was then sealed with an aluminum gasket containing a PTFE/silica gel septum. In the course of e-nose analysis, crimp-top vials were warmed at 40 °C for 30 min in a dry bath heater. Headspace gas (2 mL) was injected into the Fox 4000 system and pumped into the sensor chamber with a constant rate of 150 mL∙min2. 2-octanol (0.7 mL∙min−1) was added as an internal standard before the vials were sealed. The vials were placed into a SPME autosampler , coupled to an Agilent 7890A gas chromatograph (GC) and 5975C mass spectrometer (MS). Volatile analysis was performed using a DB-WAX column . Helium was used as a carrier gas at 1.0 mL∙min−1. The temperature programme was set as follows: it started at 40 °C and was increased by 3 °C∙min−1 to 100 °C and then to 245 °C at 5 °C∙min−1. The column effluent was ionized by electron ionization (EI) at an energy of 70 eV with a transfer temperature of 250 °C and a source temperature of 230 °C. Volatiles were identified by comparison with the mass spectra of the National Institute of Standards and Technology (NIST) Mass Spectral Library (NIST-08) or with authentic standards when available . Relative quantification of compounds was carried out using the peak area of the internal standard as a reference based on total ion chromatogram (TIC).Volatile compounds were extracted using solid phase micro extraction (SPME) as previously reported . Approxihttp://www.metaboanalyst.ca/) with default parameter. PLS-DA model validation was performed by permutation test in which p < 0.05. VIP values exceeding 1.0 were selected as cutoff. Data were normalized using ‘Autoscaling’ in the metaboAnalyst program. The pearson correlation between e-nose sensors response and volatile content was analyzed using Origin 8.0 . A completely randomized design was used in the experiments. Standard errors were calculated by Microsoft Excel. Principle component analysis (PCA) and discriminant function analysis (DFA) are used for volatiles components detected by e-nose using AlphaSoft version 11.0 . MULTI EXPERIMENT VIEWER (version 4.6.0) was applied for heatmaps of volatiles. Content of volatile compounds was subjected to partial least squares discriminant analysis (PLS-DA), and the value of variable importance in projection (VIP) was conducted by MetaboAnalyst 3.0  of the 18 sensors (S1–S18) for peach samples are shown in a polar plot in To detect potential differences among volatiles produced by different samples of peach fruit juice, PCA and DFA were used to check this capability of e-nose in assigning peach fruit samples to a specific group. PCA analysis showed that the PC1 and PC2 explained over 97.3% of the total variation in the peach color-volatiles relationship , indicatPLS-DA is a classification technique based on partial least squares (PLS). The general principle is that the regression model between the independent variable X and the categorical variable Y was established based on the information of sample classification in the process of variables extraction, and then the samples were classified according to the PLS prediction value of samples. In this study, the PLS-DA model was established between the encoded binary variety numbers and the electronic nose data. In this process of PLS-DA, the Y values of peach samples were set 1 and −1 for red peach and white peach, respectively, and then the regression of Y onto X was calculated. To further test the capability of e-nose in the classification of two different colored peach samples, the model of PLS-DA was carried out through analyzing the e-nose data. It was worth noting that the calibrated PLS model must be evaluated for its validity, and the validation method of segmented cross-validation was applied for the validation purpose. In the process of segmented cross-validation, several samples were used as the calibration set, and the remaining samples were used as the validation set, until all samples were validated. The process is looped until all samples were validated. In this study, all peach samples were carried out for calibration and validation. The accuracy of modeling set and calibration set in the PLS-DA classification model was 97.9% and 97.9%, respectively. The results indicate that it is possible to use the PLS-DA classification model to classify the red and white peaches by analyzing the volatile compounds measured by e-nose. Multivariate analysis of the response of the e-nose sensors showed that red-fleshed and white-fleshed peach samples could be classified, indicating possible difference in volatile compounds released by these two peach groups. Therefore, the traditional technique GC-MS was applied to detect difference in concentrations of individual volatile component, although this method is time-consuming, complicated, and costly. A total of 50 volatiles were detected in a total of 48 peach samples, including C6 compounds, esters, lactones, terpenes, norisopernoids, ketones, alcohols, phenypropanoids, and others. It was somewhat surprising that variations occurred in volatile contents by as much as approximately 1045-fold across peach samples . Signifip < 0.01). In PLS-DA score plot, red-fleshed peach fruit were separated from white-fleshed samples  were calculated. VIP is used to explain the weight of the independent variable in explaining the dependent variable, which is usually considered to have important roles in the PLS-DA discriminant process when values exceed 1.0. A total of 18 volatile compounds with VIP score > 1.0 were identified b, indicaE)-2-hexenal, 1-hexanol, and (Z)-2-hexen-1-ol -2-hexenal, 2-hexenal, 1-hexanol, and (Z)-2-hexen-1-ol. Descriptive sensory analysis revealed that the unpleasant flavor of peach fruit was highly associated with β-damascenone [Volatiles were used to create a classification function that distinguished the Calanda peach cultivar from other cultivars in Europe . In the ascenone , which hascenone ,10. AlthPresent observations showed that both e-nose and GC-MS analysis could be applied to detect the difference in volatile profiles between peach cultivars due to flesh color. The application of e-nose provided an overview volatile profile of fruits and improved the effectiveness and the efficiency of the process. The progress of e-nose application in fruit aroma evaluations was reviewed, and the potential for future development were proposed . GC-MS aIn summary, peach fruit with different flesh colors varies in volatile profiles at ripe stages. E-nose revealed different sensory qualities between white- and red-fleshed peach fruit, and GC-MS unraveled at least 18 volatiles that contribute to flavor difference. Red-fleshed peach fruit accumulated more fruity or peach like volatiles compared to that produced by white-fleshed fruit. Volatiles showed different concentrations in white- and red-fleshed peach, such as lactones and terpenoids. Candidate genes contributed to differences in volatile profiles between peach fruit with different colors that are required to be characterized in future.